Cloning And Expression Of Lipase Genes 7 And 8 From Yarrawia Lipolytica

It has been reported there have 8 genes of lipase in Yarrwia lipolytica. Cloning and expression of lipase gene 7 (lip7) and lipase gene 8 (lip8) from Y. lipolytica created a platform for comparative study of Y.lipolytica lipases family, and for development and application of the lipases. Based on the bioinformatics, two pairs of primers were designed according to the nucleotide sequence of AJ549519 and AJ549520, and lip7, lip8 were directly cloned by PCR. Nucleotide sequencing revealed that the lip7 and lip8 respectively had an ORF of 1101 bp and 1116 bp corresponding to 366aa and 371aa. The deduced amino acid sequence alignment through BLAST revealed 100% and 99% identity to the reported LIP7 and LIP8, respectively. The genes from Y.lipolytica were cloned into pPIC9K expression vector, and expressed in Pichia pastoris GS115 for the first time. Cultures of recombined P. pastoris accumulated active enzymes in the supernatant to levels of 49.49U/L and 35.98U/L after inducing for 96 hours in flask,respectively. The molecular masses of the recombinant lipase protein (YLL7 and YLL8) determined by SDS-PAGE were both closed to 40KDa.The optimal temperature and pH for lipolytic activity of the YLL7 were 55℃and 8.0, respectively. It retained 29% activity when kept in 65℃for 2 hours. The lipase was highly stable in the pH 6.0~8.0 and retained 75% of its original activity for 4hrs. The enzyme activity was strongly stimulated by Mg2+, and slightly inhibited by Cu2+. The optimum temperature and pH for lipolytic activity of the Y. lipolitia lipase 8 were 45℃and 8.0, respectively. It was stable at temperature below 50℃, and the lipase was highly stable in pH 6.0~8.0 and retained 90%of its original activity for 4hrs. The enzyme activity was strongly stimulated by Zn2+, and slightly inhibited by Cu2 +, Mn2+. Y. lipolitia lipase 7 and 8 had a maximal activity for medium chain esters ester p-nitrophenyl-C4~C8.The primary structure, the secondary structure, and the tertiary structure of the YLL7 and YLL8 were annotated for the first time through the homologous lipase with known structure in BioEdit software, PSIPRED server and SwissModel server, respectively. The results show that Y. lipolytica lipases 7 and 8 are conserved in GHSLG region. LIP7 has an active center for the catalytic triad comprising of Ser192, Asp258, His271, and contains 32.34% ofα-helex, 14.56% ofβ-folded, 53.10% of other structures, Ala114, Asp115, Ala116, Ile117 form a "lid" covering on the active sites. While LIP8 contains 31.15% of α-helix, 16.94% ofβsheet and 51.91% of the other structures, and its active center is composed of Ser192, Asp258, His318.